Micro RNAs (miRNAs) are the smallest, functional, non-coding RNAs of plants and animals. To date over 200 miRNAs have been discovered across all multicellular phylogeny including C. elegans, Drosophila, plants, mice and humans. The mature miRNA is approximately 22 nt in length and is cleaved from partially duplexed precursors (termed pre-miRNA) by the ribonuclease III Dicer. Although over 100 human miRNAs have been discovered thus far, very little is known about their targets, levels of expression and function. Recently, two human miRNAs (miR15 and miR16) have been mapped to a region (13q14) that is commonly deleted in chronic lymphocytic leukemia (CLL). Northern blotting demonstrated that the expression of miR15 and miR16 was reduced in CLL patients with loss of heterozygosity (LOH) at 13q14. The purpose of this proposal is to develop sensitive assays to quantify miRNAs in clinical specimens. The long-term goal of this work is to investigate if a link exists between the expression of human miRNAs and the development of cancer. Since miRNAs are different from traditional RNAs they present distinct challenges as far as their quantification. Pre-miRNA exists as a stable hairpin and the mature miRNA is roughly the size of a standard PCR primer. We propose PCR methods to overcome these obstacles and permit sensitive, high-throughput quantification of this new class of RNAs. A real-time, quantitative PCR assay will be developed to screen the expression of all known 100+ human pre- and mature miRNAs. The real-time PCR data will be presented in a micro array format using a cluster algorithm to compare the relative ratios of the pre-miRNA. The assays will be tested and validated on cDNA from human tumor cell lines. The expression of miR15 and miR16 pre- and mature miRNAs will be measured in a pilot study of 40 CLL patients. The expression of both miRNAs will be correlated to the LOH at 13q14 in these patients. We will screen the expression of over 100 human pre-miRNAs in samples of normal, benign, primary and metastatic prostate tissue from 60 patients. We propose to develop sensitive, high-throughput methodologies to screen the expression of an entirely new class of genes that have only been discovered within the past two years. Since these are very small, noncoding RNAs, their expression is not possible using commercially available microarrays. Assays developed with this research may help to identify the function of a new class of genes in humans.